Biological Activities and the Essential Oil Analysis of Cousinia harazensis and C. calocephala

This research aimed to evaluate the cytotoxicity, anti-bacterial, anti-fungal and heme polymerization inhibition activities, as well as the detection of the chemical composition of essential oils and measurement of the amount of total phenol and flavonoids of Cousinia harazensis and C. calocephala. In-vitro growth inhibitory effects of methanol extracts on A2780, T-47D, A549 and Hep-G2 cells were evaluated by MTT assay. MIC and MBC/MFC were determined by the agar dilution method. The anti-malarial activity of herbs was assessed with an inhibition test of heme detoxification (ITHD). Total phenol and flavonoids content measured by Folin-Ciocalteu method. The essential oils from two herbs were extracted by hydro-distillation, and GC/MS analyzed their compositions. Cell studies against selected cell lines growth in MTT assay were related to C. harazensis on Hep-G2 with IC50 of 4.521 µg/mL. The MIC of anti-bacterial and anti-fungal effects is related to C. harazensis extract on Staphylococcus epidermidis and Aspergillus fumigatus with 15.62 and 62.5 mg/mL, respectively. Both extracts do not have anti-malarial activity. C. harzensis content was richer in total phenol and flavonoids rather than the other herb. m-benzyl benzyl alcohol (46.7%) and butyl phthalate (14.7%) are the major compounds of C. harazensis; main components of C. calocephala are 3-methyl-tetrahydrofuran (24.6%) and oleic acid (15.4%). In conclusion, C. harazensis with more phenol and flavonoids content showed better results in terms of biological activities.


Introduction
Herbs are a valuable source of various novel pharmacognostic compounds. Despite some studies, the biological effects of many plants' extracts and essential oils have not yet been examined, and new species should be screened to discover new pharmacological effects (1).
Flora of Iran is one of the richest floras globally and has 1800 endemic species from 8000 taxa. The genus Cousinia with 600-700 species, is the largest genera of flowering plants in Central and Western Asia (2). After Astragalus, Cousinia with approximately 240 species, is considered as the second largest genus in flora of Iran (3). Cousinia harazensis Rech.f. (CH) and C. calocephala Jaub. & Spach (CC) are endemic to Iran (4). There are few studies about biological activities and phytochemical research of the genus Cousinia, thus, in this article, some biological effects of the herbs were studied.
Regarding the side effects, high toxicity, and drug resistance observed with current anticancer and anti-microbial agents, especially synthetic drugs, many kinds of research have been oriented toward natural components, especially from plants; so in this work, we studied cytotoxicity, anti-bacterial, anti-fungal and anti-malarial activities and essential oil analysis of CC and CH for the first time.
Despite the advances in biomedical research and innovative technologies, cancer is a growing public health problem; the estimated number of new cancer cases in the United States in 2020 is over 1.8 M (5). One study in 2019 showed that chemical components of C. davisiana, C. foliosa, C. ramosissima and C. stenocephala were examined against Colo205 (human colon carcinoma) and A549 (human non-small cell lung cancer) cell lines. The best result was related to C. stenocephala (IC 50 for Colo205: 130 and for A549: 990 (μg/mL)) and ψ-taraxasterol (IC 50 for Colo205: ≥100 and for A549: ≥100 (μg/mL)) (6). Sajjadi et al. studied the cytotoxic activity of C. verbascifolia against OVCAR-3 and HT-29 cancer cells. The IC 50 values against OVCAR-3 and HT-29 cells were 119 -190 and 118 -194 μg/mL, respectively (7). Sajjadi et al., in another study, isolated two phenolic components from the C. verbascifolia that had moderated cytotoxicity against OVCAR-3 and HT-29 cell lines (8).
Infections-induced death is one of the ten first mortality factors in the world (10). Many new anti-microbial and anti-parasite agents have been discovered and marketed. Still, because of the genetic ability to change gene expression and other mechanisms, resistance to these drugs is growing (11,12).
The importance of resistance to antimicrobial and anti-parasite drugs has not only emerged in hospitals but also occurred in community environments (11,12) and it can be so harmful, especially for patients with the suppressed immune system. According to new infections, it can lead to an increase in hospitalization (13).
Methanol extracts of C. lachnosphaera affect Pseudomonas aeruginosa and Staphylococcus aureus; and C. turkmenorum against S. aureus (14). Anti-bacterial activity of C. onopordioides, C. rechingerae, C. hypopolia, C. sulabadensis, C. phyllocephala, C. smirnovii and C. verbascifolia were assessed against five selected bacteria (15). Amiri et al. studied anti-bacterial activity from the root of C. microcarpa against some selected Gram-positive and Gram-negative bacteria. The best result of the disk diffusion method was for methanol extract with a 19 mm zone against P. aeruginosa (16).
In anti-fungal activity, Bahraminejad et al. evaluated a methanol extract of C. stenocephala and found that this extract cannot inhibit the growth of Fusarium oxysporum (17). In another study, none of the selected species of Cousinia were reported to have activity against Candida albicans (14). Bahraminejad et al. assessed the anti-fungal potency of C. stenocephala against Phytophtora drechsleri, Pythium aphanidermatum and Rhizoctonia solani and the results showed that the methanol extract has acceptable effects (18).
However, no studies have addressed the anti-malarial activity of Cousinia, and the present study is the first assessment of inhibition test of heme detoxification from the species.
Essential oils are in some higher plants, and they have some applications such as carminative, flavor agents, anti-microbial activities, etc. (19,20). This research aims to render information to report the oil constituents of CH and CC.

Plant Materials
Aerial parts of CH and CC were collected from Lasem Area (Mazandaran Province, Iran) in July 2018. Voucher specimens were deposited in the Central Herbarium of Tehran University, Tehran, Iran, by No: 48400 and 48401, respectively.
A. Rastegar A. identified the herbs at Kurdistan Agriculture and Natural Resources Research and Education Center, Sanandaj, Iran. The herbs were cleaned and dried, and then they were grounded to a fine powder.

Extraction Procedure
One-hundred grams of powdered parts of each herb were macerated in 300 mL methanol 80% (Chem-Lab, Belgium), and they were shaken for 24 h (GFL, Germany) at room temperature and repeated (X 3). Then, solvents were evaporated using a rotary evaporator (Heidolph, Germany). The concentrated extracts were dried in dry-oven at 40 °C to remove the remained solvents. The resulting extracts were kept in sterile containers at 4 °C for further tests.

MTT assay
As mentioned before, after 48 h, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium containing MTT was then aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. Then, the plates were gently agitated until the formed formazans were dissolved. The absorbance of each well was read using a plate reader (Rainbow, Australia) at 570 nm, subtracting the absorbance at 650 nm as a reference (there is no absorbance by MTT at 570 nm). Sample groups were compared by ANOVA with Bonfferoni correction, and nonlinear regression was applied to estimate the IC 50 of each extract.

Determination of Inhibition Zone
Pre-evaluation of the anti-bacterial activity of the extracts was studied by the cup-plate method. In short, bacterial suspension with turbidity equal to 0.5 McFarland standards (1 × 10 8 CFU/mL) was prepared. The MHA plates were streaked by this suspension. The wells with diameters 8 mm were filled with 100 μL of different concentrations of the extracts, including 500, 250, 125, 62.5 mg/mL. The solvent was also added in a well as a negative control. The plates were incubated at 37°C for 18-24 h. The cup-plate method was performed 3 times, and the average diameters of inhibition zones for each concentration were determined.

Determination of Minimum Inhibitory Concentration (MIC)
MIC was determined by the micro-

Determination of Minimum Bactericidal Concentration (MBC)
Twenty microliters of each well, in which no growth was added to MHA medium and plates were incubated at 37 °C for 18-24 h.

Fungal Strains and Culture Media Candida albicans ATCC 10231 and
Aspergillus fumigatus PTCC 5009 were purchased from IROST, Tehran, Iran.
C. albicans was cultured on Sabouraud Dextrose Agar (SDA) medium and incubated at 37 °C for 24-48 h. A. fumigatus was activated at 27 °C for 24-48 h in a similar medium and condition.

Preparation of Fungal Suspensions
Candida albicans (yeast) Standard suspension yeast was prepared by broth micro-dilution method as described by Clinical and Laboratory Standards Institute (CLSI) guidelines, document M27-S3 (23). The RPMI 1640 medium (Gibco, USA) was used. The optical turbidity of C. albicans suspension was adjusted to 75 to 77% at a wavelength of 530 nm, and 1000 times more dilutions were performed with the mentioned medium. The final inoculum concentration was adjusted to 0.5 × 10 3 to 2.5 × 10 3 CFU/ mL (24).

Aspergillus fumigatus (Mold)
To prepare a standard suspension, CLSI guideline, document M38-A2 was used, and the optical turbidity of this mold suspension was adjusted to 78 to 82% at a wavelength of 530 nm, 50 more dilutions were done with RPMI 1640, and final inoculum concentration was adjusted to make about 5 × 10 4 CFU/mL (25).

Determination of Inhibition Zone
Pre-evaluation of anti-fungal activities of these methanol extracts was performed via the disk diffusion test and based on the CLSI method (26). 100 μL of each standard suspension was added on the surface of the plates filled with SDA medium and then speared in three roundabouts with a sterile loop. Then, the disks with diameters equal to 6.4 mm were used. These disks were smeared with different concentrations of the extracts, including 500, 250, 125, 62.5 mg/mL dissolved in distilled water and tween 80 (D/T) (8:2), D/T was used as a negative control. All plates were incubated for 24-48 h. The disk diffusion method was performed 3 times, and the average diameters of inhibition zones for each concentration were determined.

Determination of Minimum Inhibitory Concentration (MIC)
To evaluate the MIC value based on CLSI methods, CH was mixed with RPMI 1640 and Tween 80 (80:20) and this solvent was used as a negative control and CC was mixed completely with RPMI 1640 to make homogeneous concentrations: 500, 250. 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.95, 0.97 and 0.48 mg/mL (One row was considered for evaluation of each extract sterility). One-hundred microliters of each standard suspension was added to each well of microplate. One column without plant extract was considered as growth control. The plates with C. albicans suspension were incubated at 27 °C, and plates that contained A. fumigatus were incubated at 37 °C for 24-48 h (X 2).

Determination of Minimum Fungicidal Concentration (MFC)
Ten μL of each well content in which no growth observed was added to the SDA medium, and plates were incubated at mentioned conditions.

Inhibition Test of Heme Detoxification
Inhibition of β-hematin formation was used for the inhibition test of heme detoxification as a spectrophotometric assay. Hemin Chloride (Merck, Germany) was dissolved in DMSO (Merck, Germany). The solution was diluted (1:1) freshly with 1 M acetate buffer (pH 4.8). Diluted hemin (60 μg/mL -1 ), tween 20 (0.012 g/L), and samples (200 μg.mL -1 ) were mixed in each well of a 96-well plate with a ratio of 9:9:2, respectively. The herbs and chloroquine diphosphate were used as negative and positive controls, respectively. The plates were incubated at 60 °C for 24 h (X 3). Finally, the absorbance was recorded with an ELISA reader at 405 nm. The results were calculated as the percentage of heme detoxification inhibition (27).

Total phenolic content assay
Total amounts of phenolic compounds in each extract were determined by Folin-Ciocalteu colorimetric method. A calibration curve obtained with Gallic acid (Merck, Germany) as a standard was drawn, and phenolic compounds in the extracts were carried out in triplicate and calculated according to this line diagram equation. Total phenolic contents were expressed as a percentage of Gallic acid equivalents in dry extract matter (28).

Total flavonoid content assay
The total amount of flavonoid compounds was assessed by the AlCl 3 colorimetric method (28). A calibration curve obtained with rutin (Merck, Germany) as a standard was drawn, and flavonoid compounds in the extracts were carried out in triplicate and calculated according to this line diagram equation. Total flavonoid contents were expressed as a percentage of rutin equivalents in dry extract matter.

Isolation of the Essential Oil
The essential oils were obtained by the hydro-distillation method by using a Clevenger-type apparatus for 3 h. The essential oils were dried over anhydrous sodium sulfate and kept in a sealed dark vial at 4 °C until analysis.

GC-MS Analysis
GC-MS analysis was carried out on a Thermofinnigan GC-MS instrument equipped with a DB-5 fused silica column (30 m X 0.5 µm, film thickness 0.33 mm). The oven temperature program was raised from 50 °C to 100 °C at a rate of 5 °C/min, and then raised 100 °C to 150 °C for 2 min at a rate of 5 °C/ min; then set up 150 °C to 200 °C for 2 min at a rate of 5 °C/min; then raised 200 °C to 230 °C for 2 min at a rate of 5 °C/min and then raised 230 °C to 260 °C for 2 min at a rate of 10 °C/min and held for 2 min. Helium was used as the carrier gas at a flow rate of 1.5 ml/ min; The splitless mode was performed. The quadrupole mass spectrometer was scanned over the 35-375 AMU with an ionizing voltage of 70 eV and an ionization current of 150 mA.
The essential oil components were identified by comparing their mass spectra with those reported in the literature and presented in the internal reference mass spectra library (Wiley/NIST).

Yield of Extraction
The extraction yields of Cousinia harazensis and C. calocephala were 10.6 and 10.4%, respectively.

Analysis of Essential Oils
The essential oils were colorless and characteristic odor. Analysis of oil from CH and CC identified 9 and 15 compounds, representing 98.9 and 63.9% of the total constituents, respectively. The identified compounds and their percentage are summarized in Tables 1 and 2. m-benzyl benzyl alcohol (46.7%), butyl phthalate (14.7%), oleic acid (14.2%) and palmitic acid (11.2%) are some of the major compounds of CH; main components of CC are 3-methyl-tetrahydrofuran (24.6 %) and oleic acid (15.4%).
The main class of the compounds in herbs is presented in Table 3. According to this table, CC has monoterpenes and sesquiterpenes (10.4 and 5.9%).

Evaluation of Cytotoxicity
Different results from two extracts against A2780, T-47D, A549 and Hep-G2, were obtained. According to the results of CH, apart from the effect of methanol extract against A2780 that was relative and transient proliferative (RTP effect), other results showed that the best cytotoxic effect was related to the effect of CH against Hep-G2 (IC 50 : 4.52 ± 3.19 µg/mL, p < 0.05).
Methanol extract of CC has no effects on T-47D and A549, but CC has a proliferative effect against A2780, and the EC 50 of it was 8.03 ± 4.49 µg/mL. All MTT assays are presented in Table 4. According to Balantyne et al. about the classification of the cytotoxicity for natural ingredients, based on this article, the result of CH against Hep-G2 is considered as a potentially very toxic extract. The effect of CH on T-47D and A549 might be classified as potentially toxic (29).      Table 3. Category of major components in CH and CC (%).

Zone of Inhibition Diameters
Pre-evaluations of anti-bacterial activity of the samples using the cup-plate technique are presented in Tables 5 and 6. These results showed that the diameters of inhibition zones of the samples dilutions were increased concerning the concentration of the extracts except in CH. Results showed that in the concentration of 500 mg/ml against P. aeruginosa, the diameter of the inhibition zone was decreased. The best result of this item was related to the effect of CH (Conc. 500 mg/mL) against S. epidermidis with 31.3 ± 0.70 mm inhibition zone diameter.

Result of MIC Value
In order to determine the MIC values, twofold serial dilutions from concentrations of 500 to 0.48 mg/mL were used. Between both Gram-positive and Gram-negative strains, CH inhibits in lower concentration than CC. The lowest concentration that could inhibit the growth of bacteria was related to CH extract on S. epidermidis with a 15.62 mg/mL MIC value ( Table 7).

Result of MBC Value
According to Table 8, both extracts can kill Gram-positive bacteria in the same concentrations; however, by comparing the effect of these two extracts on Gram-negative bacteria, it has been found that CH has the best results, especially its effect on P. aeruginosa and S. enterica with MBC value 62.5 mg/mL.

Anti-Fungal Results Zone of Inhibition Diameters
Pre-evaluation of anti-fungal activity of the extracts was performed by using disk Hep-G2 IC50: 4.52 ± 3.19 RTP effect Table 4. The results of cytotoxicity by MTT assay method (µg/mL), numbers represent mean ± SD.  Table 5. The mean (±SD) of inhibition zone diameter of extracts in Gram-positive bacteria (mm), n = 3.  Table 6. The mean (±SD) of inhibition zone diameter of extracts in Gram-negative bacteria (mm), n = 3. diffusion technique. These results showed that the extracts in each concentration could not inhibit the growth of fungal strains.

Result of MIC Value
In order to determine the MIC values, two serial dilutions from the concentration of 500 to 0.48 mg/mL were used (Table 9). CH had the most anti-fungal effect against A. fumigatus with MIC 62.5 mg/mL.

Result of MFC Value
Similar MIC results are obtained for MFC, and CH proved better than CC. The results are shown in Table 10.

Result of Inhibition Test of Heme Detoxification (ITHD)
In this method, if the percentage of ITHD was more than 90%, the assay would be considered as positive, and values less than 90% indicated a negative result. The results of the two extracts do not have anti-malarial activity in this mechanism.

The Results of Total Phenol and Flavonoid content assays
The total phenol compounds was evaluated    A. fumigatus 62.5 500 Table 9. The mean MIC of extracts in different fungal (mg/mL), n = 2.  by Folin-Ciocalteu colorimetric method, which showed that CH and CC were 7.45 and 4.57%, total flavonoid content assays were present 4.57 and 2.73%, respectively.

Discussion
In this study, we assessed cytotoxicity, anti-bacterial, anti-fungal and inhibition test of heme detoxification method for antimalarial activities of CH and CC. Assessment of anti-malarial activity of Cousinia species was studied for the first time.
According to the results of the MTT assay, different cytotoxicity and proliferative effects were seen from these extracts. Except for A2780, the results of the cytotoxicity effect of CH were significant against T-47D, A549 and Hep-G2. Despite the result of CH on these cell lines, CC did not show any effect on T-47D and A549. In contrary, CH presented a proliferative effect on A2780 and RTP effect on the Hep-G2 cell lines.
The result of our study on CH on A549 is comparable with C. davisiana, C. foliosa, C. ramosissima and C. stenocephala (6) that has shown the highest cytotoxicity for CH than others. No other investigations were found to compare our results for the herbal extracts against these cell lines.
About the result of the anti-bacterial and anti-fungal activities of two methanol extracts, the results show that CH is more effective than another extract. Both CH and CC had better effects on selected bacteria strains rather than C. lachnosphaera and C. turkmenorum (14). These results show that CH can inhibit all of the Gram-positive and Gram-negative bacteria. However, C. lachnosphaera affects P. aeruginosa and S. aureus, while C. turkmenorum was only effective on S. aureus. None of the extracts in these two studies can inhibit the growth of C. albicans.
Amiri et al. studied the anti-bacterial activity of C. microcarpa (16). Looking at the inhibition zone diameter results of his study, CH with 31.5 mm has the best effects against P. aeruginosa compared to C. microcarpa with 19 mm.
To our knowledge, few studies been have been conducted on the chemical compositions of Cousinia species so far. According to Tables 1 and 2, two compounds, including 1-methoxy-4-(1-methoxy phenyl) benzene and m-benzyl benzyl alcohol, are similar in structure. The other compound, ethyl linoleate was found in the essential oil of Daphne sericea (3.9%), Gardenia jasminoides (1.3%) and Datura metel (17.4%), too (30)(31)(32). Some other compounds, including oleic acid, palmitic acid and stearic acid were detected in Centaurea solstitialis, Moringa oleifera and Citrus spp. peel (33)(34)(35). α-pinene, as we reported 5.3% in CH, is one of the famous compounds, and it is found in turpentine very much (36). This compound has different biological effects such as anti-peptic ulcer, anti-malarial and antibacterial activity (37)(38)(39). According to Tekin et al., fatty acids/fatty acid esters (46.1%) and esters (20.2%) are the integral ingredients of C. savasica, and the results indicated the similarity between the class of compounds of C. savasica and C. harazensis with varying components (40).
In conclusion, the biological research indicates that C. harazensis has considerable potential and justifiable of folk and traditional use. The biological effects can make contributions to the isolation of active ingredients. These effects can be attributed to their phenol and flavonoids contents.